The echinocandins (ECs) are a new antifungal group that includes caspofungin, micafungin, and anidulafungin. ECs represent a profoundly important development in antifungal chemotherapy because they are generally fungicidal, non-toxic, and remain active versus strains that acquire resistance to other antifungals. These advantages stem from the unique mechanism ofEC action involving inhibition of fungal p-l ,3-glucan synthase and hence cell wall synthesis. Reduced echinocandin susceptibility (RES) can develop in normally susceptible fungi such iIS Candida albicans by mutation of FKSl encoding the major glucan synthase, but fOrtw'l ately such mutations occur only rarely. However, a number ofless common but often more lethal fungal pathogens demonstrate intrinsic RES of varying levels. FusarIUm, Scedosporium, Cryptococcus, Trichosporon, and zygomycete s~ies are characterized by high-level intrinsic RES, while Aspergillus specics and Candida parapsilosis exhibit low-level intrinsic RES. Intrinsic RES represents a serious limitation to EC use, since antifungal treatment is often empirical and antifungaJ prophylaxis is increasingly relied upon in high risk patients. To address this limitation, we must understand the basis for intrinsic RES. Our central hypothesis is that intrinsic RES, as in mutationally acquired RES, is mediated by Fks protein sequences. Support for this hypothe;is has been provided by Our recently published studies examining intrinsic resistance ofC. paraosilosis (GarciaEffron et al .. 200S. Me 52:2305) and Fusariwn solani (Katiyar and &i1ind. 2009. MC in press). We propose to fyrfug tcst this by using the yeast Saccharomyces cerevisiae as model and surrogate host, through the following Aims: (I) Mutatiorlally defIne the EC target in S. cerevisiae FksJ. Begirlning with our recently expanded database of spontaneous RES mutations (manuscripts in preparation). this Aim will rely on a new method for peR-based site-direcled mutagenesis (pSM) that is efficient, versatile... and immune to ex"l)ression artifacts. Differential efferu of mlltation~ on ca.<;JlOfUrWn.. micafungin, and anidulat\ltlgln susceptibility will be used to deduce the EC binding site. These data will be correlated with Fht topology, ftM PSM used to refine these models. (2) Examine the bflsis for intrinsic RES of selected fungal pathogens (ScedQsrorium , Cryp(QCQccus. and Aspergillus species) by hetaologous FKS expression in S. cerevisiae. Sequence aligrunents coupled with Aim 1 data will identity candidate RES-mediating regions. These will be used to generate fu l hybrids in S. ceevi.siae using the PSM method, followed. by tests ofEC susceptibility. Finally, PSM will be used to test the predicted roles of specific Fks residue;in intrinsic RES. These studies will accelerate the development of second generation ECs that provide a broader spectrum of protection from fungal pathogens.